U.S. patent application number 10/031123 was filed with the patent office on 2004-11-11 for modified human granulocyte-colony stimulating factor and process for producing same.
Invention is credited to Bae, Sung-Min, Jung, Sung-Youb, Kwon, Se-Chang, Lee, Gwan-Sun.
Application Number | 20040224393 10/031123 |
Document ID | / |
Family ID | 36129291 |
Filed Date | 2004-11-11 |
United States Patent
Application |
20040224393 |
Kind Code |
A1 |
Kwon, Se-Chang ; et
al. |
November 11, 2004 |
Modified human granulocyte-colony stimulating factor and process
for producing same
Abstract
A modified human granulocyte-colony stimulating factor(hG-CSF)
is produced by culturing a microorganism transformed with an
expression vector comprising a gene encoding a modified hG-CSF to
produce and secrete the modified hG-CSF to periplasm, said modified
hG-CSF being obtained by replacing at least one of the 1st, 2nd,
3rd and 17th amino acids of wild-type hG-CSF(SEQ ID NO: 2) with
other amino acid
Inventors: |
Kwon, Se-Chang; (Seoul,
KR) ; Jung, Sung-Youb; (Seoul, KR) ; Bae,
Sung-Min; (Seoul, KR) ; Lee, Gwan-Sun; (Seoul,
KR) |
Correspondence
Address: |
David A. Einhorn
ANDERSON, KILL & OLICK, P.C.
1251 Avenue of the Americas
New York
NY
10020-1182
US
|
Family ID: |
36129291 |
Appl. No.: |
10/031123 |
Filed: |
January 9, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10031123 |
Jan 9, 2002 |
|
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|
PCT/KR00/00733 |
Jul 7, 2000 |
|
|
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Current U.S.
Class: |
435/69.5 ;
435/320.1; 435/325; 530/351; 536/23.5 |
Current CPC
Class: |
C07K 2319/034 20130101;
C12N 15/625 20130101; C07K 2319/75 20130101; A61P 35/02 20180101;
C07K 14/535 20130101; C12N 15/70 20130101 |
Class at
Publication: |
435/069.5 ;
435/320.1; 435/325; 530/351; 536/023.5 |
International
Class: |
C07H 021/04; C12P
021/02; C07K 014/535 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 8, 1999 |
KR |
1999/27418 |
Claims
What is claimed is:
1. A modified human granulocyte-colony stimulating factor(hG-CSF)
which is characterized in that at least one of the 1st, 2nd, 3rd
and 17th amino acids of wild-type hG-CSF(SEQ ID NO: 2) is replaced
by other amino acid(s).
2. The modified hG-CSF of claim 1 whose amino acid sequence is the
same as that of wild-type hG-CSF, except that (a) the 1st amino
acid is Ser; (b) the 1st amino acid is Ser and the 17th amino acid
is X; (c) the 2nd amino acid is Met and the 3rd amino acid is Val;
(d) the 2nd amino acid is Met, the 3rd amino acid is Val and the
17th amino acid is X; or (f) the 17th amino acid is X, wherein X is
an amino acid which is not charged at neutral pH.
3. The modified hG-CSF of claim 2, wherein X is Ser, Thr, Ala or
Gly.
4. The modified hG-CSF of claim 3, wherein X is Ser.
5. A DNA encoding the modified hG-CSF of any one of claims 1 to
4.
6. The DNA of claim 5, wherein the 1st to the 60th nucleotide
sequence of the modified hG-CSF DNA corresponds to one selected
from the group consisting of SEQ ID NOS: 55, 57, 59, 61, 63, 65, 67
and 69.
7. An expression vector comprising the DNA of claim 5.
8. The expression vector of claim 7, which further comprises a
polynucleotide encoding a signal peptide attached at the 5'-end of
the DNA encoding the modified hG-CSF.
9. The expression vector of claim 8, wherein the signal peptide is
E. coli thermoresistant enterotoxin II signal peptide or modified
E. coli thermoresistant enterotoxin II signal peptide.
10. The expression vector of claim 9, wherein the E. coli
thermoresistant enterotoxin II signal peptide has the amino acid
sequence of SEQ ID NO: 53.
11. The expression vector of claim 9, wherein the modified E. coli
thermoresistant enterotoxin II signal peptide has the amino acid
sequence of SEQ ID NO: 54.
12. The expression vector of claim 9, which further comprises a
modified E. Coli enterotoxin II Shine-Dalgano sequence having the
nucleotide sequence of SEQ ID NO: 71.
13. The expression vector of claim 8, wherein the signal peptide is
E. coli beta lactamase signal peptide or modified E. coli beta
lactamase signal peptide.
14. The expression vector of claim 13, wherein the E. coli beta
lactamase signal peptide has the amino acid sequence of SEQ ID NO:
24.
15. The expression vector of claim 8, wherein the signal peptide is
E. coli Gene III signal peptide or modified E. coli Gene III signal
peptide.
16. The expression vector of claim 15, wherein the E. coli Gene III
signal peptide has the amino acid sequence of SEQ ID NO: 42.
17. The expression vector of claim 7 or 8, which is pT14SS1SG,
pT14SS1S17SEG, pTO1SG, pTO1S17SG, pTO17SG or pBAD2M3V17SG.
18. A microorganism transformed with the expression vector
according to claim 7 or 8.
19. The microorganism of claim 18, which is a transformed E.
coli.
20. The microorganism of claim 19, wherein the transformed E. coli
is E. coli BL21(DE3)/pT14SS1SG(HM 10310), E. coli
BL21(DE3)/pT14SS1S17SEG(HM 10311, KCCM-10154), E. coli
BL21(DE3)/pTO1SG(HM 10409), E. coli BL21(DE3)/pTO1S17SG(HM 10410,
KCCM-10151), E. coli BL21(DE3)/pTO17SG(HM 10411, KCCM-10152), E.
coli BL21 (DE3)/pTO17TG(HM 10413), E. coli BL21 (DE3)/pTO17AG(HM
10414), E. coli BL21(DE3)/pTO17GG(HM 10415), E. coli
BL21(DE3)/pBAD2M3VG(HM 10510, KCCM-10153), E. coli
BL21(DE3)/pBAD17SG(HM 10511) or E. coli BL21(DE3)/pBAD2M3V 17SG(HM
10512).
21. A process for producing a modified hG-CSF in microorganism
which comprises culturing the transformed microorganism of claim 18
to produce and secrete the modified hG-CSF to periplasm. E. coli
thermoresistant enterotoxin II signal peptide or modified E. coli
thermoresistant enterotoxin II signal peptide.
10. The expression vector of claim 9, wherein the E. coli
thermoresistant enterotoxin II signal peptide has the amino acid
sequence of SEQ ID NO: 53.
11. The expression vector of claim 9, wherein the modified E. coli
thermoresistant enterotoxin II signal peptide has the amino acid
sequence of SEQ ID NO: 54.
12. The expression vector of claim 9, which further comprises a
modified E. coli enterotoxin II Shine-Dalgano sequence having the
nucleotide sequence of SEQ ID NO: 71.
13. The expression vector of claim 8, wherein the signal peptide is
E. coli beta lactamase signal peptide or modified E. coli beta
lactamase signal peptide.
14. The expression vector of claim 13, wherein the E. coli beta
lactamase signal peptide has the amino acid sequence of SEQ ID NO:
24.
15. The expression vector of claim 8, wherein the signal peptide is
E. coli Gene III signal peptide or modified E. coli Gene III signal
peptide.
16. The expression vector of claim 15, wherein the E. coli Gene III
signal peptide has the amino acid sequence of SEQ ID NO: 42.
17. The expression vector of claim 7 or 8, which is pT14SS1SG,
pT14SS1S17SEG, pTO1SG, pTO1S17SG, pTO17SG or pBAD2M3V17SG.
18. A microorganism transformed with the expression vector
according to claim 7 or 8.
19. The microorganism of claim 18, which is a transformed E. coli.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation patent application of PCT
Patent Application No. PCT/KR00/00733, which was filed on Jul. 7,
2000, designating the United States of America, now abandoned.
FIELD OF THE INVENTION
[0002] The present invention relates to a modified human
granulocyte-colony stimulating factor(hG-CSF), a gene encoding said
peptide, a vector comprising said gene, a microorganism transformed
with said vector and a process for producing the modified hG-CSF
using said microorganism.
BACKGROUND OF THE INVENTION
[0003] The term colony stimulating factor (CSF) is inclusive of
granulocyte/macrophage-colony stimulating factor(GM-CSF),
macrophage-colony stimulating factor(M-CSF) and granulocyte-colony
stimulating factor(G-CSF), which are produced by T-cells,
macrophages, fibroblasts and endothelial cells. GM-CSF stimulates
stem cells of granulocyte or macrophage to induce the
differentiation thereof and proliferation of granulocyte or
macrophage colonies. M-CSF and G-CSF primarily induce the formation
of the colonies of macrophage and granulocyte, respectively. In
vivo, G-CSF induces the differentiation of bone marrow leucocytes
and enhances the function of mature granulocyte and, accordingly,
it's clinical importance in treating leukemia has been well
established.
[0004] Human G-CSF(hG-CSF) is a protein consisting of 174 or 177
amino acids, the 174 amino-acid variety having higher
neutrophil-enhancing activity(Morishita, K. et al., J. Biol. Chem.,
262, 15208-15213(1987)). The amino acid sequence of hG-CSF
consisting of 174 amino acids is shown in FIG. 1 and there have
been many studies for the mass production of hG-CSF by manipulating
a gene encoding said hG-CSF.
[0005] For instance, Chugai Pharmaceuticals Co., Ltd.(Japan) has
disclosed the amino acid sequence of hG-CSF and a gene encoding
same(Korean Patent Publication Nos. 91-5624 and 92-2312), and
reported a method for preparing proteins having hG-CSF activity by
a gene recombination process(Korean Patent Nos. 47178, 53723 and
57582). In this preparation method, glycosylated hG-CSF is produced
in a mammalian cell by employing a genomic DNA or cDNA comprising a
polynucleotide encoding hG-CSF. The glycosylated hG-CSF has an
O-glycosidic sugar chain, but, it is known that said sugar chain is
not necessary for the activity of hG-CSF(Lawrence, M. et al.,
Science, 232, 61(1986)). Further, it is also well-known that the
production of glycosylated hG-CSF employing mammalian cells
requires expensive materials and facilities, and therefore, such a
process is not economically feasible.
[0006] Meanwhile, there have been attempts to produce
non-glycosylated hG-CSF by employing a microorganism, e.g., E.
coli. In these studies, hG-CSFs having 175 or 178 amino acids
having a methionine residue attached at the N-terminus thereof are
obtained due to the ATG initiation codon employed in the
microorganism. The additional methionine residue, however, causes
undesirable immune responses in human body when the recombinant
hG-CSF is administered thereto(European Patent Publication No.
256,843). Further, most of the methionine-containing hG-CSFs
produced in E. coli are deposited in the cells as insoluble
inclusion bodies, and they must be converted to an active form
through a refolding process, at a significant loss of yield. In
this regard, four of the five Cys residues present in wild-type
hG-CSF participate in forming disulfide bonds, while the remaining
one contributes to the aggregation of the hG-CSF product during the
refolding process to lower the yield.
[0007] Recently, in order to solve the problems associated with the
production of a foreign protein within a microbial cell, various
efforts have been made to develop a method based on efficient
secretion of a target protein across the microbial cell membrane
into the extra-cellular domain.
[0008] For instance, in a method employing a signal peptide, a
desired protein is expressed in the form of a fusion protein
wherein a signal peptide is added to the N-terminus of the protein.
When the fusion protein passes through the cell membrane, the
signal peptide is removed by an enzyme and the desired protein is
secreted in a mature form. The secretory production method is
advantageous in that the produced amino acid sequence is usually
identical to the wild-type. However, the yield of a secretory
production method is often quite low due to unsatisfactory
efficiencies in both the membrane transport and the subsequent
purification process. This is in line with the well-known fact that
the yield of a mammalian protein produced in a secretory mode in
prokaryotes is very low: Hitherto, no microbial method has been
reported for the efficient expression and secretion of soluble
hG-CSF having no added methionine residue at its N-terminus.
[0009] The present inventors have previously reported the use of a
new secretory signal peptide prepared by modifying the signal
peptide of E. coli thermoresistant enterotoxin II(Korean Patent
Laid-open publication No. 2000-19788) in the production of hG-CSF.
Specifically, an expression vector comprising a hG-CSF gene
attached to the 3'-end of the modified signal peptide of E. coli
thermoresistant enterotoxin II was prepared, and biologically
active, mature hG-CSF was expressed by employing E. coli
transformed with the expression vector. However, most of the
expressed hG-CSF accumulated in the cytoplasm rather than in the
periplasm.
[0010] The present inventors have endeavored further to develop an
efficient secretory method for the production of hG-CSF in a
microorganism and have found that a modified hG-CSF, which is
prepared by replacing at least one amino acid residue, especially,
the 17th cysteine residue, of wild-type hG-CSF with other amino
acid, retains the biological activity of the wild-type, and that
the modified hG-CSF having no methionine residue at the N-terminus
thereof can be efficiently expressed and secreted by a
microorganism when an appropriate secretory signal peptide is
employed.
SUMMARY OF THE INVENTION
[0011] Accordingly, it is an object of the present invention to
provide a modified human granulocyte-stimulating factor(hG-CSF)
which can be efficiently produced using a microorganism.
[0012] It is another object of the present invention to provide a
gene encoding said peptide and a vector comprising said gene.
[0013] It is a further object of the present invention to provide a
microorganism transformed with said vector.
[0014] It is a still further object of the present invention to
provide a process for producing a hG-CGF which is non-attached
methionine residue to amino terminus using said microorganism.
[0015] In accordance with one aspect of the present invention,
there is provided a modified hG-CSF characterized in that at least
one of the 1st, 2nd, 3rd and 17th amino acids of wild-type hG-CSF
is replaced by another amino acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The above and other objects and features of the present
invention will become apparent from the following description of
the invention taken in conjunction with the following accompanying
drawings; which respectively show:
[0017] FIG. 1: the nucleotide and amino acid sequences of wild-type
human granulocyte-stimulating factor composed of 174 amino acids
residues(SEQ ID NOS: 1 and 2);
[0018] FIG. 2: the procedure for constructing vector pT-CSF;
[0019] FIG. 3: the procedure for constructing vector pT14S1SG;
[0020] FIG. 4: the procedure for constructing vector pT14SS1SG;
[0021] FIG. 5: the procedure for constructing vector
pT140SSG-4T22Q;
[0022] FIG. 6: the procedure for constructing vector
pT14SS1S17SEG;
[0023] FIG. 7: the procedure for constructing vector pTO1SG;
[0024] FIG. 8: the procedure for constructing vector PBADG;
[0025] FIG. 9: the procedure for constructing vector pBAD2M3VG;
[0026] FIGS. 10a and 10b: the results of western blot analyses
which verily the expression of hG-CSF and modified hG-CSFs from
recombinant cell lines and the molecular weight of expressed
proteins, respectively; and
[0027] FIG. 11: the cellular activities of hG-CSF and modified
hG-CSF produced from recombinant cell lines.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The modified hG-CSFs of present invention are derived by
replacing one or more of the amino acids of wild-type hG-CSF(SEQ ID
NO: 2), preferably the 1st, 2nd, 3rd and 17th amino acids thereof,
by other amino acids. More preferred are those obtained by
replacing the 17th amino acid of hG-CSF with an amino acid which is
uncharged at neutral pH. Specific examples of preferred modified
hG-CSFs have the amino acid sequence of wild-type hG-CSF, except
that:
[0029] (a) the 1st amino acid is Ser;
[0030] (b) the 1st amino acid is Ser and the 17th amino acid is
X;
[0031] (c) the 2nd amino acid is Met and the 3rd amino acid is
Val;
[0032] (d) the 2nd amino acid is Met, the 3rd amino acid is Val and
the 17th amino acid is X; or
[0033] (f) the 17th amino acid is X,
[0034] wherein X is an amino acid which is not charged at neutral
pH., preferably Ser, Thr, Ala or Gly, more preferably Ser.
[0035] Four of the five Cys residues of hG-CSF participate in
forming disulfide bonds, while the 17th Cys residue remains
unbonded in the natural state. However, when a large amount of
hG-CSF is expressed in recombinant cells, the 17th Cys residue gets
involved in inter-molecular disulfide bond formation, leading to
the accumulation of agglomerated hG-CSFs in the cytoplasm. However,
the inventive modified hG-CSF having an amino acid other than Cys
at the 17th position is free of such problem and can be effectively
produced by a secretory method using an appropriately transformed
microorganism.
[0036] The modified hG-CSF of the present invention may be encoded
by a gene comprising a nucleotide sequence deduced from the
modified hG-CSF amino acid sequence according to the genetic code.
It is known that several different codons encoding a specific amino
acid may exist due to the codon degeneracy, and, therefore, the
present invention includes in its scope all nucleotide sequences
deduced from the modified hG-CSF amino acid sequence. Preferably,
the modified hG-CSF gene sequence includes one or more preferred
codons of E. coli.
[0037] The gene thus prepared may be inserted to a conventional
vector to obtain an expression vector, which may, in turn, be
introduced into a suitable host, e.g., an E. coli. The expression
vector may further comprise a signal peptide. Representative signal
peptides include a thermoresistant E. coli. enterotoxin II signal
peptide(SEQ ID NO: 53), a modified thermoresistant E. coli
enterotoxin II signal peptide(SEQ ID NO: 54), a beta lactamase
signal peptide(SEQ ID NO: 24), Gene III signal peptide(SEQ ID NO:
42) or modified peptide thereof, but these do not limit the signal
peptides which may be used in the present invention. The promoter
used in preparing the expression vector of present invention may be
any of those which can express a heterologous protein in a
microorganism host. Specially, lac, Tac, and arabinose promoter is
preferred when the heterologous protein is expressed from E.
coli.
[0038] Exemplary expression vector of the present invention
includes pT14SS1SG, pT14SS1S17SEG, pTO1SG, pTO1S17SG, pTO17SG,
pTO17TG, pTO17AG, pTO17GG, pBAD2M3VG, pBAD17SG and
pBAD2M3V17SG.
[0039] The expression vectors of the present invention may be
introduced into microorganism, e.g., E. coli BL21(DE3)(Novagen), E.
coli XL-1 blue(Novagen) according to a conventional transformation
method(Sambrook et al., the supra) to obtain transformants E. coli
BL21(DE3)/pT14SS1SG(HM 10310), E. coli BL21(DE3)/pT14SS1S17SEG(HM
10311), E. coli BL21(DE3)/pTO1SG(HM 10409), E. coli
BL21(DE3)/pTO1S17SG(HM 10410), E. coli BL21(DE3)/pTO17SG(HM 10411),
E. coli BL21(DE3)/pTO17TG(HM 10413), E. coli BL21(DE3)/pTO17AG(HM
10414), E. coli BL21 (DE3)/pTO17GG(HM 10415), E. coli
BL21(DE3)/pBAD2M3VG(HM 10510), E. coli BL21(DE3)/pBAD17SG(HM 10511)
and E. coli BL21(DE3)/pBAD2M3V17SG(HM 10512). Among the transformed
microorganism, preferred are transformants E. coli
BL21(DE3)/pT14SS1S17SEG(HM 10311), E. coli BL21(DE3)/pTO1S17SG(HM
10410), E. coli BL21(DE3)/pTO17SG(HM 10411) and E. coli
BL21(DE3)/pBAD2M3VG(HM 10510) which were deposited with Korean
Culture Center of Microorganisms(KCCM)(Address; Department of Food
Engineering, College of Eng., Yonsei University, Sodaemun-gu, Seoul
120-749, Republic of Korea) on Mar. 24, 1999 under accession
numbers KCCM-10154, KCCM-10151, KCCM-10152 and KCCM-10153,
respectively, in accordance with the terms of the Budapest Treaty
on the International Recognition of the Deposit of Microorganism
for the Purpose of Patent Procedure.
[0040] The modified hG-CSF protein of th present invention may be
produced by culturing the transformant microorganism to express the
gene encoding the modified hG-CSF protein and secrete the modified
hG-CSF, protein to periplasm; and recovering the modified hG-CSF
protein from the periplasm. The transformant microorganism may be
cultured in accordance with a conventional method(Sambrook et al.,
the supra). The microorganism culture may be centrifuged or
filtered to collect the microorganism secreting the modified hG-CSF
protein. The transformed microorganism may be disrupted according
to a conventional method(Ausubel, F. M. et al., Current Protocols
in Molecular Biology, (1989)) to obtain a periplasmic solution. For
example, the microorganism may be disrupted in a hypotonic
solution, e.g., distilled water, by an osmotic shock. Recovery of
the modified hG-CSF in the periplasmic solution may be conducted by
a conventional method(Sambrook et al., the supra), e.g., ion
exchange chromatography, gel filtration column chromatography or
immune column chromatography. For example, hG-CSF may be purified
by sequentially conducting CM-Sepharose column chromatograph and
Phenyl Sepharose column chromatography.
[0041] The modified hG-CSF protein produced according to the
present invention is not methionylated at the N-terminus and has
biological activity which is equal to, or higher than, that of
wild-type hG-CSF. Therefore, it may be used as is in various
applications
[0042] The following Examples are intended to further illustrate
the present invention without limiting its scope.
EXAMPLE 1
Preparation of A Gene Encoding hG-CSF
[0043] A cDNA gene encoding hG-CSF was prepared by carrying out PCR
using as an hG-CSF template(R&D system, USA). The primers used
are those described in U.S. Pat. No. 4,810,643.
[0044] To prepare a cDNA gene encoding mature hG-CSF, vector
pUC19-G-CSF(Biolabs, USA) was subjected to PCR using the primers of
SEQ ID NOS: 3 and 4. The primer of SEQ ID NO: 3 was designed to
provide an NdeI restriction site(5'-CATATG-3') upstream from the
first amino acid(threonine) codon of mature hG-CSF, and the primer
of SEQ ID NO: 4, to provide a BamHI restriction site(5'-GGATCC-3')
downstream from the termination codon thereof.
[0045] The amplified hG-CSF gene was cleaved with NdeI and BamHI to
obtain a gene encoding mature hG-CSF. The hG-CSF gene was inserted
at the NdeI/BamHI section of vector pET14b(Novagen, USA) to obtain
vector pT-CSF.
[0046] FIG. 2 shows the above procedure for constructing vector
pT-CSF.
EXAMPLE 2
Construction of a Vector Containing the Gene Encoding E. coli
Enterotoxin II Signal Peptide and a Modified hG-CSF
[0047] (Step 1) Cloning E. coli Enterotoxin II Signal Peptide
Gene
[0048] To prepare E. coli enterotoxin II signal peptide gene, the
pair of complementary oligonucleotides having SEQ ID NOS: 5 and 6
were designed based on the nucleotide sequence of E. coli
enterotoxin II signal peptide, and synthesized using DNA
synthesizer(Model 380B, Applied Biosystem, USA).
[0049] The above oligonucleotides were designed to provide BspHI
restriction site(complementary sites to an NcoI restriction sites)
upstream from the initiation codon of E. coli enterotoxin II and an
MluI restriction site introduced by a silent change at the other
end.
[0050] Both oligonucleotides were annealed at 95.degree. C. to
obtain blunt-ended DNA fragments having a nucleotide sequence
encoding E. coli enterotoxin II signal peptide(STII gene).
[0051] The STII gene was inserted at the SmaI site of vector
pUC19(Biolabs, USA) to obtain vector pUC 19ST.
[0052] (Step 2) Preparation of a Gene Encoding STII/hG-CSF
[0053] To prepare a gene encoding STII/hG-CSF, vector pT-CSF
obtained in Preparation Example 1 was subjected to PCR using the
primers of SEQ ID NOS: 7 and 8. The primer of SEQ ID NO: 7 was
designed to substitute Ser codon for the first codon of hG-CSF, and
the primer of SEQ ID NO: 8, to provide a BamHI restriction
site(5'-GGATCC-3') downstream from the termination codon
thereof.
[0054] The amplified DNA fragments were cleaved with MluI and
BamHI, and then inserted at the MluI/BamHI section of pUC19ST
obtained in Step 1 to obtain vector pUC19S1SG. Vector pUC19S1SG
thus obtained contained a gene encoding an STII/hG-CSF(designated
STII-hG-CSF gene).
[0055] Vector pUC-19S1SG was cleaved with BspHI and BamHI to obtain
a DNA fragment(522 bp). The DNA fragment was inserted at the
NcoI/BamHI section of vector pET14b(Novagen, USA) to obtain vector
pT14S1SG.
[0056] FIG. 3 depicts the above procedure for constructing vector
pT14S1SG.
[0057] (Step 3) Addition of E. coli Enterotoxin II Shine-Dalgarno
Sequence to STII-hG-CSF Gene
[0058] Vector pT14S1SG obtained in Step 2 was subjected to PCR
using the primers of SEQ ID NOS: 9 and 10. The primer of SEQ ID NO:
9 was designed to provide an E. coli enterotoxin II Shine-Dalgano
sequence(designated STII SD sequence) and an XbaI restriction site,
and the primer of SEQ ID NO: 10, to provide a BamHI restriction
site downstream from the termination codon of mature hG-CSF to
obtain a DNA fragment(STII SD-STII-hCSF) containing a STII SD and
STII-hG-CSF gene.
[0059] The STII SD-STII-hG-CSF fragment was cleaved with XbaI and
BamHI, and then inserted at the XbaI/BamHI section of vector
pET14b(Novagen, USA) to obtain vector pT14SS1SG.
[0060] FIG. 4 displays the above procedure for constructing vector
pT14SS1SG.
[0061] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pT14SS1SG to obtain a transformant designated E. coli HM
10310.
[0062] (Step 4) Construction of a Vector Containing a Gene Encoding
STII/hG-CSF Fusion Protein
[0063] The first codon of the modified hG-CSF gene of plasmid
pT14SS1SSG obtained in Step 3 was replaced by Thr in accordance
with a site-directed mutagenesis(Papworth, C. et al., Strategies,
9, 3(1996)), which was conducted by PCR of the plasmid with a sense
primer(SEQ ID NO: 12) having a modified nucleotide sequence, a
complementary antisense primer(SEQ ID NO: 13), and pfu(Stragene,
USA).
[0064] The amplified DNA fragment was recovered and restriction
enzyme DpnI was added thereto to remove unconverted plasmids.
[0065] E. coli XL-1 blue(Novagen, USA) was transformed with the
modified plasmid. The base sequence of the DNA recovered from
transformed colonies was determined, and thus obtained was plasmid
pT14SSG which contained a gene having Thr in place of the first
amino acid of hG-CSF(SEQ ID NO: 11).
1 -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 Thr Asn Ala Tyr Ala Thr Pro Leu Gly
Pro (SEQ ID NO: 11) - ACA-AAT-GCC-TAC-GCG-ACA-CCC-CTG-GGC-CCT (SEQ
ID NO: 12) - TGT-TTA-CGG-ATG-CGC-TGT-GGG-GAC-CCG-GGA (SEQ ID NO:
13)
[0066] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pT14SSG to obtain a transformant designated E. coli HM
10301.
[0067] (Step 5) Construction of a Vector Containing a Gene Encoding
Modified STII/hG-CSF
[0068] Vector pT14SSG obtained in Step 4 was subjected to PCR using
the complementary primers of SEQ ID NOS: 15 and 16, which were
designed to substitute Thr codon for the 4th codon of STII in
accordance with the procedure of Step 4 to obtain a modified
plasmid.
[0069] E. coli XL-1 blue(Novagen, USA) was transformed with the
modified plasmid. The base sequence of the DNA recovered from
transformed colonies was determined, and thus obtained was plasmid
which contained a gene having Thr in place of the 4th amino acid of
STII(SEQ ID NO: 14).
2 Met Lys Lys Thr Ile Ala Phe Leu (SEQ ID NO: 14)
5'-GG-TGT-TTT-ATG-AAA-AAG-ACA-ATC-GCA-TTT-CTT-C-3' (SEQ ID NO: 15)
3'-CC-ACA-AAA-TAC-TTT-TTC-TGT-TAG-CGT-AAA-GAA-G-5- ' (SEQ ID NO:
16)
[0070] The plasmid thus obtained was cleaved with XbaI and MluI,
and then inserted at the XbaI/MluI section of vector pT14SSG
obtained in step 4 to obtain vector pT14SSG-4T.
[0071] (Step 6) Construction of a Vector Containing a Gene Encoding
Modified STII/hG-CSF
[0072] Vector pT14SSG-4T obtained in Step 5 was subjected to PCR
using the complementary primers of SEQ ID NOS: 18 and 19, which
were designed to substitute Gln codon for the 22nd codon of STII in
accordance with the procedure of Step 4 to obtain a modified
plasmid.
[0073] E. coli XL-1 blue(Novagen, USA) was transformed with the
modified plasmid. The base sequence of the DNA recovered from
transformed colonies was determined, and thus obtained was plasmid
pT14SSG-4T22Q which contained a gene having Gln in place of the
22nd amino acid of STII(SEQ ID NO: 17).
3 ASN Ala Gln Ala Thr Pro Leu Gly (SEQ ID NO: 17)
5'-CA-AAT-GCC-CAA-GCG-ACA-CCC-CTG-GGC-3' (SEQ ID NO: 18)
3'-GT-TTA-CGG-GTT-CGC-TGT-GGG-GAC-CCG-5' (SEQ ID NO: 19)
[0074] (Step 7) Construction of a Vector Containing a Modified STII
SD and a Gene Encoding Modified STII/hG-CSF
[0075] Vector pT14SSG-4T22Q obtained in Step 6 was subjected to PCR
using the complementary primers of SEQ ID NOS: 20 and 21 in
accordance with the procedure of Step 4 to obtain vector
pT140SSG-4T22Q having the six nucleotide sequences between the STII
SD sequence(GAGG) and the initiation codon of STII(modified STII SD
of SEQ ID NO: 71).
[0076] FIG. 5 represents the above procedure for constructing
vector pT140SSG-4T22Q.
[0077] E. coli BL21(DE3) was transformed with vector pT140SSG-4T22Q
to obtain a transformant designated E. coli HM 10302.
EXAMPLE 3
Construction of a Vector Containing a Gene Encoding Modified
hG-CSF
[0078] To prepare a modified hG-CSF gene, S1 oligomer(SEQ ID NO:
22) having E. coli-preferred codons and Ser in place of the 17th
amino acid of hG-CSF and AS1 oligomer(SEQ ID NO: 23) were
synthesized using DNA synthesizer(Model 380B, Applied Biosystem,
USA).
[0079] 0.5 .mu.l (50 pmole) quantities of the oligonucleotides were
reacted at 95.degree. C. for 15 min. and kept until 35.degree. C.
for 3 hours. The mixture was precipitated in ethanol and subjected
to gel electrophoresis(SDS-PAGE) to obtain a cohesive ended double
strand(ds) oligomer.
[0080] The plasmid pT14SS1SG obtained in step 3 of Example 2 was
cleaved with ApaI and BstXI, and then ligated with the
adhesive-ended ds oligomer, to obtain vector pT14SS1S17SEG. Vector
pT14SS1S17SEG contained a gene encoding hG-CSF having E.
coli-preferred codons at the amino terminus and Ser in place of the
1st and 17th amino acids of hG-CSF, respectively.
[0081] FIG. 6 illustrates the above procedure for constructing
vector pT140SS1S17SEG.
[0082] E. coli BL21(DE3) was transformed with vector pT14SS1S17SEG
to obtain a transformant designated E. coli HM 10311, which was
deposited with Korean Culture Center of Microorganisms(KCCM) on
Mar. 24, 1999 under accession number KCCM-10154.
EXAMPLE 4
Construction of Vector Containing a Gene Encoding E. coli OmpA
Signal Peptide and Modified hG-CSF
[0083] A vector containing a gene encoding Tac promoter and OmpA
signal peptide(SEQ ID NO: 24) as well as a gene encoding modified
hG-CSF was prepared as follows:
4 Met-Lys-Lys-Thr-Ala-Ile-Ala-Ile-Ala-Val-Ala-Leu-Ala-Gly-Phe-Ala-
(SEQ ID NO: 24) Thr-Val-Ala-Gln-Ala- --GTT-GCG-CAA-GCT-TCT-CGA--
(SEQ ID NO: 25) --CAA-CGC-GTT-CGA-AGA-GCT-- (SEQ ID NO: 26)
[0084] Vector pT-CSF obtained in Example 1 was subjected to PCR
using a primer(SEQ ID NO: 27) designed to substitute Ser codon for
the 1st codon of hG-CSF and another primer(SEQ ID NO: 28), to
provide an EcoRI restriction site(5'-GAATTC-3') downstream from the
termination codon thereof to obtain a DNA fragment containing a
gene encoding modified hG-CSF.
[0085] The DNA fragment was cleaved with HindIII and EcoRI, and
then inserted at the HindIII/EcoRI section of vector
pFlag.CTS(Eastman, USA) to obtain vector pTO1SG which contained a
gene encoding E. coli OmpA signal peptide and modified hG-CSF(SEQ
ID NO: 29).
[0086] FIG. 7 exhibits the above procedure for constructing vector
pTO1SG. E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pTO1SG to obtain a transformant designated E. coli HM
10409.
EXAMPLE 5
Construction of a Vector Containing a Gene Encoding E. coli OmpA
Signal Peptide and Modified hG-CSF
[0087] The first codon of the modified hG-CSF gene of plasmid
pTO1SG obtained in Example 4 was replaced by Thr in accordance with
site-directed mutagenesis(Papworth, C. et al., Strategies, 9,
3(1996)), by conducting PCR of the plasmid pTO1SG obtained in
Example 4 with a sense primer(SEQ ID NO: 30) designed to substitute
Thr codon for the 1st codon of hG-CSF and a complementary antisense
primer(SEQ ID NO: 31).
[0088] E. coli XL-1 blue(Novagen, USA) was transformed with the
modified plasmid. The base sequence of the DNA recovered from
transformed colonies was determined, and thus obtained plasmid pTOG
which contained a gene having Thr in place of the first amino acid
of hG-CSF.
[0089] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pTOG to obtain a transformant designated E. coli HM
10401.
EXAMPLE 6
Production of Modified hG-CSFs
[0090] (a) Production of [Ser1, Ser17] hG-CSF
[0091] Vector pTO1SG obtained in Example 4 was subjected to PCR
using a sense primer(SEQ ID NO: 32) designed to substitute Ser
codon for the 17th codon of hG-CSF and a complementary antisense
primer(SEQ ID NO: 33) in accordance with the procedure of Step 4 of
Example 2 to obtain a modified plasmid.
[0092] E. coli XL-1 blue(Novagen, USA) was transformed with the
modified plasmid. The base sequence of the DNA recovered from
transformed colonies was determined and thus obtained was plasmid
pTO1S17SG which contained a gene having Ser in place of the 1st and
17th amino acids of hG-CSF.
[0093] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pTO1S17SG to obtain a transformant designated E. coli HM
10410, which was deposited with Korean Culture Center of
Microorganisms(KCCM) on Mar. 24, 1999 under accession number
KCCM-10151.
[0094] (b) Production of [Ser17] hG-CSF
[0095] Vector pTOG obtained in Example 5 was subjected to PCR using
a sense primer(SEQ ID NO: 32) designed to substitute Ser codon for
the 17th codon of hG-CSF and a complementary antisense primer(SEQ
ID NO: 33) in accordance with the procedure of Step 4 of Example 2
to obtain a modified plasmid.
[0096] E. coli XL-1 blue(Novagen, USA) was transformed with the
modified plasmid. The base sequence of the DNA recovered from
transformed colonies was determined, and thus obtained was plasmid
pTO17SG which contained a gene having Ser in place of the 17th
amino acid of hG-CSF.
[0097] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pTO17SG to obtain a transformant designated E. coli HM
10411, which was deposited with Korean Culture Center of
Microorganisms(KCCM) on Mar. 24, 1999 under accession number
KCCM-10152.
[0098] (c) Production of [Thr17] hG-CSF
[0099] Vector pTOG obtained in Example 5 was subjected to PCR using
a sense primer(SEQ ID NO: 34) designed to substitute Thr codon for
the 17th codon of hG-CSF and a complementary antisense primer(SEQ
ID NO: 35) in accordance with the procedure of Step 4 of Example 2
to obtain a modified plasmid.
[0100] E. coli XL-1 blue(Novagen, USA) was transformed with the
modified plasmid. The base sequences of the DNA recovered from
transformed colonies was determined, and thus obtained was plasmid
pTO17TG which contained a gene having Thr in place of the 17th
amino acid of hG-CSF.
[0101] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pTO17TG to obtain a transformant designated E. coli HM
10413.
[0102] (d) Production of [Ala17] hG-CSF
[0103] Vector pTOG obtained in Example 5 was subjected to PCR using
a sense primer(SEQ ID NO: 36) designed to substitute Ala codon for
the 17th codon of hG-CSF and a complementary antisense primer(SEQ
ID NO: 37) in accordance with the procedure of Step 4 of Example 2
to obtain a modified plasmid.
[0104] E. coli XL-1 blue(Novagen, USA) was transformed with the
modified plasmid. The base sequence of DNA recovered from
transformed colonies was determined, and thus obtained was plasmid
pTO17AG which contained a gene having Ala in place of the 17th
amino acid of hG-CSF.
[0105] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pTO17AG to obtain a transformant designated E. coli HM
10414.
[0106] (e) Production of [Gly17] hG-CSF
[0107] Vector pTOG obtained in Example 5 was subjected to PCR using
a sense primer(SEQ ID NO: 38) designed to substitute Gly codon for
the 17th codon of hG-CSF and a complementary antisense primer(SEQ
ID NO: 39) in accordance with the procedure of Step 4 of Example 2
to obtain a modified plasmid.
[0108] E. coli XL-1 blue(Novagen, USA) was transformed with the
modified plasmid. The base sequence of the DNA recovered from
transformed colonies was determined, and thus obtained was plasmid
pTO17GG which contained a gene having Gly in place of the 17th
amino acids of hG-CSF.
[0109] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pTO17GG to obtain a transformant designated E. coli HM
10415.
[0110] (f) Production of [Asp17] hG-CSF
[0111] Vector pTOG obtained in Example 5 was subjected to PCR using
a sense primer(SEQ ID NO: 40) designed to substitute Asp codon for
the 17th codon of hG-CSF and a complementary antisense primer(SEQ
ID NO: 41) in accordance with the procedure of Step 4 of Example 2
to obtain a modified plasmid.
[0112] E. coli XL-1 blue(Novagen, USA) was transformed with the
modified plasmid. The base sequence of the DNA recovered from
transformed colonies was determined, and thus obtained was plasmid
pTO17APG which contained a gene having Asp in place of the 17th
amino acids of hG-CSF.
[0113] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pTO17APG to obtain a transformant designated E. coli HM
10416.
EXAMPLE 7
Construction of a Vector Containing a Gene Encoding E. coli Gene
III Signal Peptide and Modified hG-CSF
[0114] (a) Construction of a Vector Containing a Gene Encoding
Arabinose Promoter and E. coli Gene III Signal Peptide
[0115] A vector containing a gene encoding arabinose promoter and
E. coli Gene III signal peptide(SEQ ID NO: 42) as well as a gene
encoding modified hG-CSG was prepared as follows:
5 Met-Lys-Lys-Leu-Leu-Phe-Ala-Ile-Pro-Leu-Val-Val-Pro- (SEQ ID NO:
42) Phe-Tyr-Ser-His-Ser- -TAT-AGC-CAT-AGC-ACC-ATG-GAG- (SEQ ID NO:
43) -ATA-TCG-GTA-TCG-TGG-TAC-CTC- (SEQ ID NO: 44)
[0116] NcoI restriction site Plasmid pBAD.gIIIA(Invitrogen, USA)
containing a gene encoding arabinose promoter and Gene III signal
peptide was cleaved with NcoI, and single stranded DNAs were
removed with Klenow DNA polymerase to obtain a blunt-ended double
stranded DNA, which was then cleaved with BglII to obtain a vector
fragment having both blunt end and a cohesive end.
[0117] Vector pT-CSF obtained in Example 1 was subjected to PCR
using a sense primer(SEQ ID NO: 46) having a nucleotide sequence
coding for the 2nd to the 9th amino acids of hG-CSF(SEQ ID NO: 45)
and a complementary antisense primer(SEQ ID NO: 47) in accordance
with the procedure of Step 4 of Example 2 to obtain a blunt-ended
DNA fragment containing hG-CSF gene and a BamHI restriction site in
the carboxy terminus. The fragment then was cleaved with BamHI to
obtain hG-CSF gene fragment having both a blunt end and a cohesive
end.
6 Pro Leu Gly Pro Ala Ser Ser Leu (SEQ ID NO 45) 5'
-C-CCC-CTG-GGC-CCT-GCC-AGC- (SEQ ID NO 46) TCC-CTG-3' 3'
-G-GGG-GAC-CCG-GGA-CGG-TCG- (SEQ ID NO 47) AGG-GAC-5'
[0118] The hG-CSF gene fragment as inserted into the vector
obtained above to obtain vector pBADG which contained a gene
encoding E. coli Gene III signal peptide and hG-CSF(SEQ ID NO:
48).
[0119] FIG. 8 describes the above procedure for constructing vector
pBADG.
[0120] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pBADG to obtain a transformant designated E. coli HM
10501.
[0121] (b) Production of [Met2, Val3] hG-CSF Plasmid
pBAD.gIIIA(Invitrogen, USA) was cleaved with NcoI and BglII to
obtain a fragment having two cohesive ends.
[0122] Vector pT-CSF obtained in Example 1 was subjected to PCR
using a sense primer(SEQ ID NO: 50) having a nucleotide sequence
coding for the 1st to the 9th amino acids of [Met2, Val3]
hG-CSF(SEQ ID NO: 49) and a complementary antisense primer(SEQ ID
NO: 51) in accordance with the procedure of Step 4 of Example 2 to
obtain a blunt-ended DNA fragment containing hG-CSF gene and a
BamHI restriction site in the carboxy terminus, which was then was
cleaved with NeoI and BamHI to obtain a hG-CSF gene fragment having
two cohesive ends.
7 Thr Met Val Gly Pro Ala Ser Ser Leu (SEQ ID NO: 49)
5'-TAC-GCG-TCC-ATG-GTG-GGC-CCT-GCC-AGC-TCC-CTG-3' (SEQ ID NO: 50)
3'-ATG-CGC-AGG-TAC-CAC-CCG-GGA-CGG-TCG-AGG-GAC-5' (SEQ ID NO:
51)
[0123] NcoI restriction site
[0124] The hG-CSF gene fragment was inserted into the vector
obtained above to obtain vector pBAD2M2VG contained a gene coding
E. coli Gene III signal peptide, and Met and Val in place of the
2nd and 3rd amino acids of hG-CSF(SEQ ID NO: 52), respectively.
[0125] FIG. 9 shows the above procedure for constructing vector
pBAD2M3VG.
[0126] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pBAD2M3VG to obtain a transformant designated E. coli HM
10510, which was deposited with Korean Culture Center of
Microorganisms(KCCM) on Mar. 24, 1999 under accession number of
KCCM-10153.
[0127] (c) Production of [Ser17] hG-CSF
[0128] Vector pBADG obtained in (a) was subjected to PCR using a
sense primer(SEQ ID NO: 32) designed to substitute Ser codon for
the 17th codon of hG-CSF and a complementary antisense primer(SEQ
ID NO: 33) in accordance with the procedure of Step 4 of Example 2
to obtain a modified plasmid.
[0129] E. coli XL-1 blue(Novagen, USA) was transformed with the
modified plasmid. The base sequence of the DNA recovered from
transformed colonies was determined, and thus obtained was plasmid
pBAD17SG which contained a gene having Ser in place of the 17th
amino acid of hG-CSF.
[0130] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pBAD 17SG to obtain a transformant designated E. coli HM
10511.
[0131] (d) Production of [Met2, Val3, Ser17] hG-CSF
[0132] Vector pBAD2M3VG obtained in (b) was subjected to PCR using
a sense primer(SEQ ID NO: 32) designed to substitute Ser codon for
the 17th codon of hG-CSF and a complementary antisense primer(SEQ
ID NO: 33) in accordance with the procedure of Step 4 of Example 2
to obtain a modified plasmid.
[0133] E. coli XL-1 blue(Novagen, USA) was transformed with the
modified plasmid. The base sequence of the DNA recovered from
transformed colonies was determined, and thus obtained was plasmid
pBAD2M3V17SG which contained a gene having Met, Val and Ser in
place of the 2nd, 3rd and 17th amino acids of hG-CSF,
respectively.
[0134] E. coli BL21(DE3)(Stratagene, USA) was transformed with
vector pBAD2M3V 17SG to obtain a transformant designated E. coli HM
10512.
EXAMPLE 8
Production of hG-CSF
[0135] Transformants prepared in Examples 2 to 7 were cultured in
LB medium(1% bacto-tryptone, 0.5% bacto-yeast extract and 1% NaCl)
and then incubated in the presence of an expression inducer(IPTG)
for 3 hours or cultured in the absence of IPTG more than 15 hours.
Each of the cultures was centrifuged at 6,000 rpm for 20 min. to
precipitate bacterial cells, and the precipitate was suspended in a
1/10 volume of isotonic solution(20% sucrose, 10 mM Tris-Cl buffer
solution containing 1 mM EDTA, pH 7.0). The suspension was allowed
to stand at room temperature for 30 min, and then centrifuged at
7,000 rpm for 10 min. to collect bacterial cells. The cells were
resuspended in D.W. at 4.degree. C. and centrifuged at 7,000 rpm
for 10 min. to obtain a supernatant as a periplasmic solution. The
hG-CSF level in the periplasmic solution was assayed in accordance
with ELISA method(Kato, K. et al., J. Immunol., 116, 1554(1976))
using an antibody against hG-CSF(Aland, USA), which was calculated
as the amount of hG-CSF produced per 1 l of culture. The results
are shown in Table I.
8TABLE 1 hG-CSF Level Transformant Example Expression Vector in
periplasm(mg/l) HM 10301 2(Step 4) pT14SSG 65 HM 10302 2(Step 7)
pT140SSG-4T22Q 277 HM 10310 2(Step 3) pT14SSlSG 92 HM 10311 3
pT14SSlS17SEG 1,512 HM 10401 5 pTOG 85 HM 10409 4 pTO1SG 105 HM
10410 6(a) pTO1S17SG 1,477 HM 10411 6(b) pTO17SG 1,550 HM 10413
6(c) pTO17TG 1,373 HM 10414 6(d) pTO17AG 1,486 HM 10415 6(e)
pTO17GG 1,480 HM 10416 6(f) pTO17APG 67 HM 10501 7(a) pBADG 54 HM
10510 7(b) pBAD2M3VG 69 HM 10511 7(c) pBAD17SG 937 HM 10512 7(d)
pBAD2M3V17SG 983
EXAMPLE 9
Purification of hG-CSF
[0136] Transformant E. coli HM 10411 prepared in Example 6(b) was
cultured in LB medium and the culture was centrifuged for 6,000 rpm
for 20 min. to harvest cells. The periplasmic solution was prepared
from the cells by repeating the procedure of Example 8.
[0137] The periplasmic solution was adjusted to pH 5.0 to 5.5,
adsorbed on a CM-Sepharose(Pharmacia Inc., Sweden) column
pre-equilibrated to pH 5.3, and then, the column was washed with 25
mM NaCl. hG-CSF was eluted by sequentially adding to the column
buffer solutions containing 50 mM, 100 mM and 200 mM NaCl, and
fractions containing hG-SCF were collected and combined.
[0138] The combined fractions were subjected to Phenyl
Sepharose(Pharmacia Inc., Sweden) column chromatography to obtain
[Ser17] hG-CSF having a purity of 99%.
[0139] Further, the above procedure was repeated using each of the
transformants E. coli HM 10311, HM 10409, HM 10411, HM 10413, HM
10414, HM 10415, HM 10510 and HM 10512 prepared in Examples 3, 4,
6(b), 6(c), 6(d), 6(e), 7(b) and 7(d), respectively.
[0140] Each of the purified hG-CSF fraction was subjected to sodium
dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE) to
determine the purity and approximate concentration of the hG-CSF,
and then subjected to ELISA to determine the exact hG-CSF
concentration in the periplasmic solution. Met-hG-CSF(Kirin amgen)
was used as a control.
[0141] FIG. 10a reproduces the SDS-PAGE result, wherein lane 1
shows Met-G-CSF, lane 2, the periplasmic solution of the
transformant E. coli HM 10411, and lane 3, the purified [Ser17]
hG-CSF. As can be seen from FIG. 10b, the molecular weight of
[Ser17] hG-CSF is the same as that of wild-type hG-CSF and the
periplasmic solution of the transformant E. coli HM 10411 contains
a high level of [Ser17] hG-CSF.
[0142] Further, the N-terminal amino acid sequences of hG-CSFs were
determined and the nucleotide sequences coding for the 1st to 32nd
amino acids produced using the transformants HM 10311, HM 10409, HM
10411, HM 10413, HM 10414, HM 10415, HM 10510 and HM 10512 shown in
SEQ ID NOS: 56, 58, 60, 62, 64, 66, 68 and 70, respectively. The
result shows that the modified hG-CSF produced according to the
present invention is not methionylated at N-terminus.
[0143] A nitrocellulose filter (Bio-Rad Lab,, USA) was wetted with
a buffer solution for blotting(170 mM glicine, 25 mM
Tris.about.HCl(PH 8), 20% methanol) and the proteins separated on
the gel were western blotted onto a nitrocellulose filter(Bio-Rad
Lab., USA.) for 3 hours. The filter was kept in 1% Casein for 1
hour and was washed three times with PBS containing 0.05% Tween 20.
The filter was put in a goat anti-G-CSF antibody(R&D System,
AB-214-NA, USA) solution diluted with PBS and reacted at room
temperature for 2 hours. After reaction, the filter was washed 3
times with a PBST solution to remove unreacted antibody.
Horseradish peroxidase-conjugated rabbit anti-goat IgG(Bio-Rad
Lab., USA) diluted with PBS was added thereto and reacted at room
temperature for 2 hour. The filter was washed with PBST, and a
peroxidase substance kit(Bio-Rad Lab.; USA) solution was added
thereto to develop a color reaction. The results from the above
western blotting are shown in FIG. 10b, wherein lane 1 represents a
positive control, Met-G-CSF, and lane 2, purified [Ser17] hG-CSF.
As can be seen from FIG. 10b, the molecular weight of [Ser17]
hG-CSF equals that of wild-type hG-CSF.
EXAMPLE 10
Cellular Activity of hG-CSF and Modified hG-CSF
[0144] Cell line HL-60(ATCC CCL-240 derived from the bone marrow of
a promyelocytic leukemia patient/a white 36-year-old woman) was
cultured in RPMI 1640 media containing 10% fetal bovine serum and
adjusted to 2.2.times.10.sup.5 cells/ml, followed by adding thereto
DMSO(dimethylsulfoxide, culture grade/SIGMA) to a concentration of
1.25% (v/v). 90 .mu.l of the resulting solution was added to a 96
well plate(Coming/low evaporation 96 well plate) in an amount of
2.times.10.sup.4 cells/well and incubated at 37.degree. C. under 5%
CO2 for 48 hours.
[0145] Each of the modified [Ala17] hG-CSF, [Gly 17] hG-CSF,
[Ser17] hG-CSF, and [Thr 17] hG-CSF was diluted in RPMI 1640 media
to a concentration of 500 ng/ml and then serially diluted 10 times
by 2-fold with RPMI 1640 media.
[0146] The resulting solution was added to wells at 10 .mu.l per
well and incubated at 37.degree. C. for 48 hours. As a positive
control, a commercially available hG-CSF(Jeil Pharmaceutical.).
[0147] The level of cell line increased was determined using a
commercially available CellTiter96.TM. (Cat # G4100, Promega) based
on the measured optical density at 670 nm.
[0148] As can be seen from FIG. 11, the cellular activities of the
modified hG-CSFs are the same as, or higher than of that the
positive control, wild-type hG-CSF.
[0149] While the embodiments of the subject invention have been
described and illustrated, it is obvious that various changes and
modifications can be made therein without departing from the spirit
of the present invention which should be limited only by the scope
of the appended claims.
Sequence CWU 1
1
71 1 522 DNA Homo sapiens CDS (1)..(522) 1 aca ccc ctg ggc cct gcc
agc tcc ctg ccc cag agc ttc ctg ctc aag 48 Thr Pro Leu Gly Pro Ala
Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 tgc tta gag caa
gtg agg aag atc cag ggc gat ggc gca gcg ctc cag 96 Cys Leu Glu Gln
Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 gag aag
ctg tgt gcc acc tac aag ctg tgc cac ccc gag gag ctg gtg 144 Glu Lys
Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 35 40 45
ctg ctc gga cac tct ctg ggc atc ccc tgg gct ccc ctg agc tcc tgc 192
Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 50
55 60 ccc agc cag gcc ctg cag ctg gca ggc tgc ttg agc caa ctc cat
agc 240 Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
Ser 65 70 75 80 ggc ctt ttc ctc tac cag ggg ctc ctg cag gcc ctg gaa
ggg ata tcc 288 Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu
Gly Ile Ser 85 90 95 ccc gag ttg ggt ccc acc ttg gac aca ctg cag
ctg gac gtc gcc gac 336 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
Leu Asp Val Ala Asp 100 105 110 ttt gcc acc acc atc tgg cag cag atg
gaa gaa ctg gga atg gcc cct 384 Phe Ala Thr Thr Ile Trp Gln Gln Met
Glu Glu Leu Gly Met Ala Pro 115 120 125 gcc ctg cag ccc acc cag ggt
gcc atg ccg gcc ttc gcc tct gct ttc 432 Ala Leu Gln Pro Thr Gln Gly
Ala Met Pro Ala Phe Ala Ser Ala Phe 130 135 140 cag cgc cgg gca gga
ggg gtc ctg gtt gct agc cat ctg cag agc ttc 480 Gln Arg Arg Ala Gly
Gly Val Leu Val Ala Ser His Leu Gln Ser Phe 145 150 155 160 ctg gag
gtg tcg tac cgc gtt cta cgc cac ctt gcg cag ccc 522 Leu Glu Val Ser
Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170 2 174 PRT Homo
sapiens 2 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
Leu Lys 1 5 10 15 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly
Ala Ala Leu Gln 20 25 30 Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
His Pro Glu Glu Leu Val 35 40 45 Leu Leu Gly His Ser Leu Gly Ile
Pro Trp Ala Pro Leu Ser Ser Cys 50 55 60 Pro Ser Gln Ala Leu Gln
Leu Ala Gly Cys Leu Ser Gln Leu His Ser 65 70 75 80 Gly Leu Phe Leu
Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 85 90 95 Pro Glu
Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp 100 105 110
Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro 115
120 125 Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
Phe 130 135 140 Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu
Gln Ser Phe 145 150 155 160 Leu Glu Val Ser Tyr Arg Val Leu Arg His
Leu Ala Gln Pro 165 170 3 32 DNA Artificial Sequence
Oligonucleotide primer for the N-terminal of hG-CSF 3 cgccgccata
tgacacccct gggccctgcc ag 32 4 36 DNA Artificial Sequence
Oligonucleotide primer for the C-terminal of hG-CSF 4 accgaattcg
gatcctcagg gctgcgcaag gtggcg 36 5 72 DNA Artificial Sequence
Oligonucleotide for preparing E. coli enterotoxin II signal peptide
5 tcatgaaaaa gaatatcgca tttcttcttg catctatgtt cgttttttct attgctacaa
60 atgcctacgc gt 72 6 72 DNA Artificial Sequence Oligonucleotide
for preparing E. coli enterotoxin II signal peptide 6 acgcgtaggc
atttgtagca atagaaaaaa cgaacataga tgcaagaaga aatgcgatat 60
tctttttcat ga 72 7 39 DNA Artificial Sequence Oligonucleotide
primer coding for the N-terminal of [Ser1]hG-CSF 7 acaaatgcct
acgcgtctcc cctgggccct gccagctcc 39 8 42 DNA Artificial Sequence
Oligonucleotide primer coding for the C-terminal of [Ser1]hG-CSF 8
accgaattcg gatcctcagg gctgcgcaag gtggcgtaga ac 42 9 65 DNA
Artificial Sequence Oligonucleotide primer coding for E.coli
enterotoxin II Shine-Dalgarno sequence 9 cggtttccct ctagaggttg
aggtgtttta tgaaaaagaa tatcgcattt cttcttgcat 60 ctatg 65 10 45 DNA
Artificial Sequence Oligonucleotide containing BamHI restriction
site 10 accgaattcg gatcctcagg gctgcgcaag gtggcgtaga acgcg 45 11 10
PRT Artificial Sequence Last five amino acids of E. coli
enterotoxin II signal peptide plus the 1st to the 5th amino acids
of hG-CSF 11 Thr Asn Ala Tyr Ala Thr Pro Leu Gly Pro 1 5 10 12 30
DNA Artificial Sequence Oligonucleotide for preparing [Thr1]hG-CSF
12 acaaatgcct acgcgacacc cctgggccct 30 13 30 DNA Artificial
Sequence Antisense of SEQ ID NO 12 13 agggcccagg ggtgtcgcgt
aggcatttgt 30 14 8 PRT Artificial Sequence N-terminal sequence of
E. coli enterotoxin II signal peptide having threonine as the 4th
amino acid 14 Met Lys Lys Thr Ile Ala Phe Leu 1 5 15 33 DNA
Artificial Sequence Oligonucleotide for substituting the 4th amino
acid of E. coli enterotoxin II signal peptide with threonine 15
ggtgttttat gaaaaagaca atcgcatttc ttc 33 16 33 DNA Artificial
Sequence Antisense of SEQ ID No 15 16 gaagaaatgc gattgtcttt
ttcataaaac acc 33 17 8 PRT Artificial Sequence C-terminal sequence
of E. coli enterotoxin II signal peptide having glutamine as the
22nd amino acid 17 Asn Ala Gln Ala Thr Pro Leu Gly 1 5 18 26 DNA
Artificial Sequence Oligonucleotide for substituting the 22nd amino
acid of E. coli enterotoxin II signal peptide with glutamine 18
caaatgccca agcgacaccc ctgggc 26 19 26 DNA Artificial Sequence
Antisense of SEQ ID NO 18 19 gcccaggggt gtcgcttggg catttg 26 20 24
DNA Artificial Sequence Oligonucleotide for modifying E. coli
enterotoxin II Shine-Dalgarno sequence 20 tctagaggtt gaggtgtttt
atga 24 21 24 DNA Artificial Sequence Antisense of SEQ ID NO 20 21
tcataaaaca cctcaacctc taga 24 22 66 DNA Artificial Sequence S1
oligomer having E. coli-preferred nucleotide sequence coding for
the 6th to 26th amino acids of [Ser17]hG-CSF 22 cagcctcttc
tcttccacaa tctttccttc ttaagtctct tgaacaagtt agaaagatcc 60 aaggcg 66
23 66 DNA Artificial Sequence Antisense of SEQ ID NO 22 (AS1
oligomer) 23 ccgggtcgga gaagagaagg tgttagaaag gaagaattca gagaacttgt
tcaatctttc 60 taggtt 66 24 21 PRT Escherichia coli SIGNAL (1)..
(21) E. coli OmpA signal peptide 24 Met Lys Lys Thr Ala Ile Ala Ile
Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala 20 25
18 DNA Artificial Sequence Oligonucleotide containing Hind III
recognition site 25 gttgcgcaag cttctcga 18 26 18 DNA Artificial
Sequence Antisense of SEQ ID NO 25 26 tcgagaagct tgcgcaac 18 27 39
DNA Artificial Sequence Oligonucleotide for the N-terminal of
[Ser1] hG-CSF 27 gttgcgcaag cttctcccct gggccctgcc agctccctg 39 28
39 DNA Artificial Sequence Oligonucleotide containing EcoRI
restriction site 28 accgaattct cagggctgcg caaggtggcg tagaacgcg 39
29 13 PRT Artificial Sequence E. coli OmpA signal peptide plus the
1st to the 5th amino acids of [Ser1]hG-CSF 29 Gly Phe Ala Thr Val
Ala Gln Ala Ser Pro Leu Gly Pro 1 5 10 30 30 DNA Artificial
Sequence Oligonucleotide for preparing [Thr1]hG-CSF 30 accgttgcgc
aagctacacc cctgggccct 30 31 30 DNA Artificial Sequence Antisense of
SEQ ID NO 30 31 agggcccagg ggtgtagctt gcgcaacggt 30 32 33 DNA
Artificial Sequence Oligonucleotide for preparing [Ser17]hG-CSF 32
agcttcctgc tcaagtcttt agagcaagtg agg 33 33 33 DNA Artificial
Sequence Antisense of SEQ ID NO 32 33 cctcacttgc tctaaagact
tgagcaggaa gct 33 34 33 DNA Artificial Sequence Oligonucleotide for
preparing [Thr17]hG-CSF 34 agcttcctgc tcaagacctt agagcaagtg agg 33
35 33 DNA Artificial Sequence Antisense of SEQ ID NO 34 35
cctcacttgc tctaaggtct tgagcaggaa gct 33 36 33 DNA Artificial
Sequence Oligonucleotide for preparing [Ala17]hG-CSF 36 agcttcctgc
tcaaggcctt agagcaagtg agg 33 37 33 DNA Artificial Sequence
Antisense of SEQ ID NO 36 37 cctcacttgc tctaaggcct tgagcaggaa gct
33 38 33 DNA Artificial Sequence Oligonucleotide for preparing
[Gly17]hG-CSF 38 agcttcctgc tcaagggctt agagcaagtg agg 33 39 33 DNA
Artificial Sequence Antisense of SEQ ID NO 38 39 cctcacttgc
tctaagccct tgagcaggaa gct 33 40 33 DNA Artificial Sequence
Oligonucleotide for preparing [Asp17]hG-CSF 40 agcttcctgc
tcaaggactt agagcaagtg agg 33 41 33 DNA Artificial Sequence
Antisense of SEQ ID NO 40 41 cctcacttgc tctaagtcct tgagcaggaa gct
33 42 18 PRT Escherichia coli SIGNAL (1)..(18) E. coli Gene III
signal peptide 42 Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val
Pro Phe Tyr Ser 1 5 10 15 His Ser 43 21 DNA Artificial Sequence
Oligonucleotide containing Nco I restriction site 43 tatagccata
gcaccatgga g 21 44 21 DNA Artificial Sequence Antisense of SEQ ID
NO 43 44 ctccatggtg ctatggctat a 21 45 8 PRT Artificial Sequence
The 2nd to the 10th amino acids of hG-CSF 45 Pro Leu Gly Pro Ala
Ser Ser Leu 1 5 46 25 DNA Artificial Sequence Oligonucleotide
primer coding for the 2nd to the 10th amino acids of hG-CSF plus an
additional cytosine at its 5'-end 46 ccccctgggc cctgccagct ccctg 25
47 25 DNA Artificial Sequence Antisense of SEQ ID NO 46 47
cagggagctg gcagggccca ggggg 25 48 10 PRT Artificial Sequence E.
coli Gene III signal peptide plus the 1st to the 5th amino acids of
hG-CSF 48 Phe Tyr Ser His Ser Thr Pro Leu Gly Pro 1 5 10 49 9 PRT
Artificial Sequence The 1st to the 9th amino acids of
[Met2,Val3]hG-CSF 49 Thr Met Val Gly Pro Ala Ser Ser Leu 1 5 50 33
DNA Artificial Sequence Oligonucleotide for preparing
[Met2,Val3]hG-CSF 50 tacgcgtcca tggtgggccc tgccagctcc ctg 33 51 33
DNA Artificial Sequence Antisense of SEQ ID NO 50 51 cagggagctg
gcagggccca ccatggacgc gta 33 52 10 PRT Artificial Sequence E. coli
Gene III signal peptide plus the 1st to the 5th amino acids of
[Met2,Val3]hG-CSF 52 Phe Tyr Ser His Ser Thr Met Val Gly Pro 1 5 10
53 23 PRT Escherichia coli SIGNAL (1)..(23) Thermoresistant E. coli
enterotoxin II signal peptide 53 Met Lys Lys Asn Ile Ala Phe Leu
Leu Ala Ser Met Phe Val Phe Ser 1 5 10 15 Ile Ala Thr Asn Ala Tyr
Ala 20 54 23 PRT Artificial Sequence Modified thermoresistant E.
coli enterotoxin II signal peptide 54 Met Lys Lys Thr Ile Ala Phe
Leu Leu Ala Ser Met Phe Val Phe Ser 1 5 10 15 Ile Ala Thr Asn Ala
Gln Ala 20 55 96 DNA Artificial Sequence Nucleotide sequence coding
for the 1st to 32nd amino acids of [Ser1, Ser17]hG-CSF 55 tct ccc
ctg ggc cct gcc agc tcc ctg ccc cag agc ttc ctg ctc aag 48 Ser Pro
Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15
tct tta gag caa gtg agg aag atc cag ggc gat ggc gca gcg ctc cag 96
Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20
25 30 56 32 PRT Artificial Sequence the 1st to 32nd amino acids of
[Ser1, Ser17]hG-CSF 56 Ser Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
Ser Phe Leu Leu Lys 1 5 10 15 Ser Leu Glu Gln Val Arg Lys Ile Gln
Gly Asp Gly Ala Ala Leu Gln 20 25 30 57 96 DNA Artificial Sequence
Nucleotide sequence coding for the 1st to the 32nd amino acids of
[Ser1]hG-CSF 57 tct ccc ctg ggc cct gcc agc tcc ctg ccc cag agc ttc
ctg ctc aag 48 Ser Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe
Leu Leu Lys 1 5 10 15 tgc tta gag caa gtg agg aag atc cag ggc gat
ggc gca gcg ctc cag 96 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
Gly Ala Ala Leu Gln 20 25 30 58 32 PRT Artificial Sequence the 1st
to the 32nd amino acids of [Ser1]hG-CSF 58 Ser Pro Leu Gly Pro Ala
Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 Cys Leu Glu Gln
Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 59 96 DNA
Artificial Sequence Nucleotide sequence coding for the 1st to the
32nd amino acids of [Ser17]hG-CSF 59 aca ccc ctg ggc cct gcc agc
tcc ctg ccc cag agc ttc ctg ctc aag 48 Thr Pro Leu Gly Pro Ala Ser
Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 tct tta gag caa gtg
agg aag atc cag ggc gat ggc gca gcg ctc cag 96 Ser Leu Glu Gln Val
Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 60 32 PRT
Artificial Sequence the 1st to the 32nd amino acids of
[Ser17]hG-CSF 60 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys 1 5 10 15 Ser Leu Glu Gln Val Arg Lys Ile Gln Gly
Asp Gly Ala Ala Leu Gln 20 25 30 61 96 DNA Artificial Sequence
Nucleotide sequence coding for the 1st to the 32nd amino acids of
[Thr17]hG--CSF 61 aca ccc ctg ggc cct gcc agc tcc ctg ccc cag agc
ttc ctg ctc aag 48 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys 1 5 10 15 acc tta gag caa gtg agg aag atc cag ggc
gat ggc gca gcg ctc cag 96 Thr Leu Glu Gln Val Arg Lys Ile Gln Gly
Asp Gly Ala Ala Leu Gln 20 25 30 62 32 PRT Artificial Sequence the
1st to the 32nd amino acids of [Thr17]hG--CSF 62 Thr Pro Leu Gly
Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 Thr Leu
Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 63
96 DNA Artificial Sequence Nucleotide sequence coding for the 1st
to the 32nd amino acids of [Ala17]hG-CSF 63 aca ccc ctg ggc cct gcc
agc tcc ctg ccc cag agc ttc ctg ctc aag 48 Thr Pro Leu Gly Pro Ala
Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 gcc tta gag caa
gtg agg aag atc cag ggc gat ggc gca gcg ctc cag 96 Ala Leu Glu Gln
Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 64 32 PRT
Artificial Sequence the 1st to the 32nd amino acids of
[Ala17]hG-CSF 64 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys 1 5 10 15 Ala Leu Glu Gln Val Arg Lys Ile Gln Gly
Asp Gly Ala Ala Leu Gln 20 25 30 65 96 DNA Artificial Sequence
Nucleotide sequence coding for the 1st to the 32th amino acids of
[Gly17]hG-CSF 65 aca ccc ctg ggc cct gcc agc tcc ctg ccc cag agc
ttc ctg ctc aag 48 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys 1 5 10 15 ggc tta gag caa gtg agg aag atc cag ggc
gat ggc gca gcg ctc cag 96 Gly Leu Glu Gln Val Arg Lys Ile Gln Gly
Asp Gly Ala Ala Leu Gln 20 25 30 66 32 PRT Artificial Sequence the
1st to the 32th amino acids of [Gly17]hG-CSF 66 Thr Pro Leu Gly Pro
Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 Gly Leu Glu
Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 67 96
DNA Artificial Sequence Nucleotide sequence coding for the 1st to
the 32nd amino acids of [Met2, Val3]hG-CSF 67 aca atg gtc ggc cct
gcc agc tcc ctg ccc cag agc ttc ctg ctc aag 48 Thr Met Val Gly Pro
Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 tgc tta gag
caa gtg agg aag atc cag ggc gat ggc gca gcg ctc cag
96 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln
20 25 30 68 32 PRT Artificial Sequence the 1st to the 32nd amino
acids of [Met2, Val3]hG-CSF 68 Thr Met Val Gly Pro Ala Ser Ser Leu
Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 Cys Leu Glu Gln Val Arg Lys
Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 69 96 DNA Artificial
Sequence Nucleotide sequence coding for the 1st to the 32nd amino
acids of [Met2, Val3, Ser17]hG-CSF 69 aca atg gtc ggc cct gcc agc
tcc ctg ccc cag agc ttc ctg ctc aag 48 Thr Met Val Gly Pro Ala Ser
Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 tct tta gag caa gtg
agg aag atc cag ggc gat ggc gca gcg ctc cag 96 Ser Leu Glu Gln Val
Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 70 32 PRT
Artificial Sequence the 1st to the 32nd amino acids of [Met2, Val3,
Ser17]hG-CSF 70 Thr Met Val Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe
Leu Leu Lys 1 5 10 15 Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
Gly Ala Ala Leu Gln 20 25 30 71 10 DNA Artificial Sequence Modified
Shine-Dalgarno sequence 71 gaggtgtttt 10
* * * * *